Reagents and methods for modulating cone photoreceptor activity

ABSTRACT

The present invention provides reagents and methods for modulating cone photoreceptor activity, and devices for assessment of cone photoreceptor activity.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/242587 filed Sep. 15, 2009, incorporated by reference hereinin its entirety.

STATEMENT OF U.S. GOVERNMENT SUPPORT

This work was supported by the National Institutes of Health grantsR01EY016861, R01EY11123, and P30EY01730. The U.S. government has certainrights in the invention.

BACKGROUND

Classic visual deprivation experiments have led to the expectation thatneural connections established during development would notappropriately process an input that was not present from birth.Therefore, it was believed that treatment of congenital vision disorderswould be ineffective unless administered to the very young.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides methods for cone cellgene therapy in a primate, comprising administering to the eye of aprimate in need of cone cell gene therapy a recombinant gene deliveryvector comprising:

(a) a promoter region, wherein the promoter region is specific forretinal cone cells; and

(b) a gene encoding a therapeutic, wherein the gene is operativelylinked to the promoter region;

wherein in vivo expression of the therapeutic in cone cells of theprimate serves to treat the primate in need of cone cell gene therapy.

The method of this aspect of the invention can be used, for example, totreat a cone cell disorder, including but not limited to colorblindness, blue cone monochomacy, achromatopsia, incompleteachromatopsia, rod-cone degeneration, retinitis pigmentosa (RP), maculardegeneration, cone dystrophy, blindness, Stargardt's Disease, andLeber's congenital amaurosis. In one embodiment, the methods restorevisual capacity in the primate; in another embodiment, the primate isable to visualize new colors as a result of the therapy. In anotherembodiment, the primate has a vision disorder in which itsphotoreceptors are healthy. In a further embodiment, the primate is anadult primate.

In another aspect, the present invention provides isolated nucleic acidexpression vector comprising:

(a) a promoter region, wherein the promoter region is specific forprimate retinal cone cells; and

(b) a gene encoding a therapeutic, wherein the gene is operativelylinked to the promoter region. In various embodiments, the vectorsfurther comprise an enhancer element upstream of the promoter, whereinthe gene is operatively linked to the enhancer element, and/or an introncomprising a splice donor/acceptor region, wherein the intron is locateddownstream of the promoter region and is located upstream of the gene.The vectors can be used, for example, in the methods of the invention.

In another aspect, the present invention provides color multi-focalelectroretinogram (mf-ERG) comprising:

(a) an electroretinogram (ERG) comprising

-   -   (i) a recording electrode that is (A) designed for placement on        at least one of a cornea and a sclera of at least one eye of a        subject and (B) arranged to output at least one signal generated        by the at least one eye; and        -   (ii) a computing system communicatively coupled to the            recording electrode, the computing system comprising (A) at            least one processor and    -   (B) data storage containing instructions executable by the at        least one processor to carry out a set of functions, the set of        functions including processing and saving the at least one        signal generated by the at least one eye;

(b) a retinal stimulator comprising matched light sources selected fromthe group consisting of red, green, blue, and ultraviolet light sources,wherein the matched light sources are connected to the ERG and inoperation can be independently frequency modulated at rates betweenabout 1 Hz and about 60 Hz, inclusive, wherein the stimulator inoperation is capable of stimulating a retinal field of a subjectthroughout an operating radius of at least about 70 degrees;

(c) one or more constant current integrated circuit chips arranged todrive the stimulator; and

(d) a pulse-frequency modulator connected to the retinal stimulator,wherein in operation the pulse-frequency modulator is capable ofcontrolling individual stimulator segments while keeping relativespectral content of the light constant. In various preferredembodiments, the matched light sources are paired red and green lightsources;

triplets of red, green, and blue light sources; or quartets of red,green, blue, and ultraviolet light sources. In another preferredembodiment the retinal stimulator comprises a concave surface comprisinga series of trapezoidal-shaped circuit boards placed edge-to-edge,wherein the concave surface positions the matched light sources so inoperation they are held equidistantly from and pointing toward a singlefocal point where a subject's pupil can be positioned.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. rAAV2/5 vector produced functional L-opsin in primate retina. a)Molecular map; TR=terminal repeats; LCR=locus control region;PP=proximal promoter; SD/SA=splice donor/acceptor; RHLOPS=recombinanthuman L opsin cDNA; PA₁=polyadenylation signal. b) Red light Multi-focalelectroretinogram (Mf-ERG) stimulus. c) mf-ERG 40 weeks after twoinjections (yellow circles) of a mixture of L-opsin- and greenfluorescent protein (GFP)-coding viruses. Grey lines show borders ofhighest response; for comparison, inset=mfERG 16 weeks post-injection;there was no reliable signal from L-opsin, unchanged from baseline. Highresponses in far peripheral retina were measured reliably and may haveoriginated from offshoot of one of the injections. d) Fluorescencephotographs from a similar retinal area as c; grey lines from c werecopied in d. e) Confocal microscopy revealed a mosaic pattern of GFPexpression in 5-12% of cones. Because GFP-coding virus was diluted to1/3 compared to L-opsin virus, an estimated 15-36% of cones inbehaviourally tested animals express L-opsin. f) Mf-ERG from abehaviourally tested animal 70 weeks after 3 injections of L-opsinvirus.

FIG. 2. Pre-therapy colour vision and possible treatment outcomes. a)Colour vision stimuli examples. b) Pre-therapy results, monkey 1. Huestested are represented as dominant wavelengths (DWs) rather than u′, v′coordinates. If a hue could not be reliably distinguished at even thehighest saturation, the extrapolated threshold approached infinity. c)Pre-therapy results, monkey 2. d)-e), Possible experimental outcomes:Monkeys could have a relative increase in long-wavelength sensitivity,but remain dichromatic (dashed lines, d); theoretical colour spectrumappearances for a dichromat and a possible “spectral shift” are shown.Alternatively, dichromatic monkeys could become trichromatic. Resultsfrom a trichromatic female control monkey are plotted (dashed line, e;error bars=SEM and n varied from 7-11).

FIG. 3. Gene therapy produced trichromatic colour vision. a) Time courseof thresholds for the blue-green confusion colour, DW =490 nm (circles),and a yellowish colour, DW=554 nm (squares). A logarithmic scale wasused to fit high thresholds for DW=490 nm; significant improvementoccurred after 20 weeks. Enclosed data points=untreated dichromaticmonkey thresholds, DW=490 nm (triangle) and DW=554 nm (diamond). b)-c)Comparison of pre-therapy (open circles, solid line) and post-therapythresholds (solid dots, dashed line). Enclosed data points are DW=490 nmthresholds when tested against a red-violet background (DW=-499 nm);pink triangles=trichromatic female control thresholds. Error bars=SEM; nvaried from 7-11.

FIG. 4. a) The geodesic dome was created by placing trapezoidal-shapedcircuit boards edge-to-edge. This structure holds the light emittingdiodes (LEDs) so they converge on a single focal point. b) The circuitboard takes the incoming control signals from the Retis-can mf-ERG andreroutes and modifies them to work with the new dome. The most frequentintegrated circuit on the board are the constant current devices. c) Thespectral composition of the red LED. d) The spectral composition of thegreen LED. e) The spectral sensitivity curves for the human M- (solidline) and L- (dashed line) cone photoreceptors. f) The activation ofM-opsin (solid line) and the L-opsin (dashed line) in response to boththe red and green LEDs.

FIG. 5. Circles and dashed line represent the red LED out-puts inmicrowatts as a function of intensity; triangles and solid linerepresent the green LED outputs. Each data point represents an averageof 3 measurements. Error bars are three standard deviations (99.7%confidence interval). To measure linearity, r2 values were computed forboth the red and green LEDs.

FIG. 6. The signal to noise ratio (SNR) for as a function of degrees ofeccentricity for a typical (averaged) subject and for the highestsubject recorded.

FIG. 7. a) Gerbil mf-ERG data in response to the L-cone isolating redstimulus. Locations of the retina that show a large amount of activityin response to stimulation by the red LEDs are indicated in red, whilethose areas that show the lowest amount of activation are indicated inblue (see scale). Gray lines show borders of the region where the mfERGresponse was highest. b) GFP fluorescence fundus image from the gerbil,scaled to the appropriate size, overlaid on the red-light mf-ERG data.The gray lines from (a) were copied into (b) to illustrate that areas ofincreased mf-ERG response corresponded to the same locations whererobust GFP fluorescence was present. c) Red-light mf-ERG data from thesquirrel monkey, which received two injections of the virus mixture, onesuperiorly and one inferiorly. d) A montage of GFP fluorescence imagesfrom the squirrel monkey, scaled to the appropriate size, overlaid onthe mfERG data.

FIG. 8. The circles represent ERG response from LEDs that were moved ona linear path while the subject fixated forward. By 30 degrees, thesignal is already less than half. In contrast, the triangles representERG response from LEDs that were fixed on a boom and rotated so that theLEDs always pointed at the pupil. Under this experimental protocol, −3dB occurs first at 60 degrees.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides methods for cone cellgene therapy in a primate, comprising administering to the eye of aprimate in need of cone cell gene therapy a recombinant gene deliveryvector comprising:

(a) a promoter region, wherein the promoter region is specific forretinal cone cells; and

(b) a gene encoding a therapeutic, wherein the gene is operativelylinked to the promoter region;

wherein in vivo expression of the therapeutic in cone cells of theprimate serves to treat the primate in need of cone cell gene therapy.

Cone cells are photoreceptor cells in the retina of the eye thatfunction best in relatively bright light. The cone cells graduallybecome sparser towards the periphery of the retina. The methods of thepresent invention can be used for treatment of any condition that can beaddressed, at least in part, by gene therapy of retinal conephotoreceptor cells. The inventors have demonstrated effective treatmentof congenital vision disorders in adult primates, a result that iscompletely unexpected in the art.

In one preferred embodiment, the gene therapy serves to treat a conecell disorder. As used herein, a “cone cell disorder” is any disorderimpacting retinal cone cells, including but not limited to colorblindness, blue cone monochomacy, achromatopsia, incompleteachromatopsia, rod-cone degeneration, retinitis pigmentosa (RP), maculardegeneration, cone dystrophy, blindness, Stargardt's Disease, andLeber's congenital amaurosis.

The gene encoding a therapeutic to be expressed in the cone cells cancomprise or consist of any gene or cDNA that encodes a polypeptide orRNA-based therapeutic (siRNA, antisense, ribozyme, shRNA, etc.) that canbe used as a therapeutic for treating a cone cell disorder. In apreferred embodiment, the primate is of the Parvorder Catarrhini. As isknown in the art, Catarrhini is one of the two subdivisions of thehigher primates (the other being the New World monkeys), and includesOld World monkeys and the apes, which in turn are further divided intothe lesser apes or gibbons and the great apes, consisting of theorangutans, gorillas, chimpanzees, bonobos, and humans. In a furtherpreferred embodiment, the primate is a human.

A “promoter” is a DNA sequence that directs the binding of RNApolymerase and thereby promotes RNA synthesis, i.e., a minimal sequencesufficient to direct transcription. Any suitable promoter region can beused in the gene therapy vectors, so long as it specifically promotesexpression of the gene in retinal cone cells. In a preferred embodiment,the promoter specifically promotes expression of the gene in primateretinal cone cells; more preferably in Catarrhini retinal cone cells;even more preferably in human retinal cone cells. As used herein,“specifically” means that the promoter predominately promotes expressionof the gene in retinal cone cells compared to other cell types, suchthat at least 80%, and preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 97%, 98%, 99%, 99.5%, or more of expression of the gene afterdelivery of the vector to the eye will be in cone cells. Exemplarysuitable promoter regions include the promoter region for anycone-specific gene, such as the L opsin promoter (SEQ ID NO:1), the Mopsin promoter (SEQ ID NO:2), and the S opsin promoter (SEQ ID NO:3), orportions thereof suitable to promote expression in a cone-specificmanner. Any suitable method for identifying promoter sequences capableof driving expression in primate cone cells can be used to identify suchpromoters, as will be understood by those of skill in the art based onthe teachings herein.

In a preferred embodiment, the gene delivery vector further comprises anenhancer element upstream of the promoter, wherein the gene isoperatively linked to the enhancer element. Enhancers are cis-actingelements that stimulate transcription of adjacent genes. Any suitableenhancer element can be used in the gene therapy vectors, so long as itenhances expression of the gene when used in combination with thepromoter. In a preferred embodiment, the enhancer element is specificfor retinal cone cells; more preferably, it is specific for primateretinal cone cells; more preferably in Catarrhini retinal cone cells;even more preferably in human retinal cone cells. As used herein,“specifically” means that the enhancer predominately enhances expressionof the gene in retinal cone cells compared to other cell types, suchthat at least 80%, and preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 97%, 98%, 99%, 99.5%, or more of expression of the gene afterdelivery of the vector to the eye will be in cone cells. Exemplarysuitable enhancer regions comprise or consist of the enhancer region forany cone-specific gene, such as the L/M minimal opsin enhancer (SEQ IDNO: 51),

L/M enhancer elements of 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, ormore nucleotides that comprise one or more copies of the L/M minimalopsin enhancer, and the full L/M opsin enhancer (SEQ ID NO:4), or otherportions thereof suitable to promote expression in a cone-specificmanner. Any suitable method for identifying enhancer sequences capableof driving expression in primate cone cells can be used to identify suchenhancers, as will be understood by those of skill in the art based onthe teachings herein.

The length of the promoter and enhancer regions can be of any suitablelength for their intended purpose, and the spacing between the promoterand enhancer regions can be any suitable spacing to promotecone-specific expression of the gene product. In various preferredembodiments, the enhancer is located 0-1500; 0-1250; 0-1000; 0-750;0-600; 0-500; 0-400; 0-300; 0-200; 0-100; 0-90; 0-80; 0-70; 0-60; 0-50;0-40; 0-30; 0-20; or 0-10 nucleotides upstream of the promoter. Thepromoter can be any suitable distance upstream of the encoded gene.

In a further preferred embodiment that can be combined with any otherembodiment in any aspect of the present invention, the enhancercomprises or consists of a sequence selected from the group consistingof the L/M minimal opsin enhancer (SEQ ID NO: 51), L/M enhancer elementsof 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, or more nucleotides thatcomprise one or more copies of the L/M minimal opsin enhancer, and thefull L/M opsin enhancer (SEQ ID NO:4), or other portions thereofsuitable to promote expression in a cone-specific manner, and thepromoter comprises or consists of a sequence selected from the groupconsisting of L opsin promoter (SEQ ID NO: 1), the M opsin promoter (SEQID NO:2), and the S opsin promoter (SEQ ID NO:3).

In a further preferred embodiment, the gene delivery vector furthercomprises an intron comprising a splice donor/acceptor region, whereinthe intron is located downstream of the promoter region and is locatedupstream of the gene. Any intron can be used, so long as it comprises asplice donor/acceptor region recognized in primate cone cells, so thatthe intron can be spliced out of the resulting mRNA product. In oneembodiment, the intron comprises or consists of an SV40 intron accordingto SEQ ID NO:5. In various preferred embodiments, the 3′ end of theintron is 0-20; 0-15; 0-10; 0-9; 0-8; 0-7; 0-6; or 0-5 nucleotidesupstream of the gene, and its 5′ end is 0-20; 0-15; 0-10; 0-9; 0-8; 0-7;0-6; or 0-5 nucleotides downstream of the proximal promoter region.

The gene is operatively linked to the promoter region and the enhancerelement, such that the promoter and enhancer elements are capable ofdriving expression of the gene or cDNA in cone cells of the subject.

The gene encoding a therapeutic to be expressed in the cone cells can beany gene or cDNA that encodes a polypeptide or RNA-based therapeutic(siRNA, antisense, ribozyme, shRNA, etc.) that can be used as atherapeutic for treating a cone cell disorder, or as a means tootherwise enhance vision, including but not limited to promotingtetrachromatic color vision. In various preferred embodiments, the geneencodes a therapeutic protein selected from the group consisting of

(a) SEQ ID NO: 7 (SEQ ID NO: 6) Homo sapiens opsin 1 (cone pigments),short-wave-sensitive (OPN1SW), mRNA NCBI Reference Sequence:NM_001708.2;

(b) SEQ ID NO: 9 (SEQ ID NO: 8) Homo sapiens opsin 1 (cone pigments),medium-wave-sensitive (OPN1MW), mRNA NCBI Reference Sequence:NM_000513.2;

(c) SEQ ID NO: 11 (SEQ ID NO: 10) Homo sapiens opsin 1 (cone pigments),long-wave-sensitive (OPN1LW), mRNA NCBI Reference Sequence:NM_(—)020061.4;

(d) SEQ ID NO: 13 (SEQ ID NO: 12) ATP binding cassette retina gene(ABCR) gene (NM_000350);

(e) SEQ ID NO: 15 (SEQ ID NO: 14) retinal pigmented epithelium-specific65 kD protein gene (RPE65) (NM_(—)000329);

(f) SEQ ID NO: 17 (SEQ ID NO: 16) retinal binding protein 1 gene (RLBP1)(NM_(—)000326);

(g) SEQ ID NO: 19 (SEQ ID NO: 18) peripherin/retinal degeneration slowgene, (NM_(—)000322);

(h) SEQ ID NO: 21 (SEQ ID NO: 20) arrestin (SAG) (NM_(—)000541);

(i) SEQ ID NO: 23 (SEQ ID NO: 22) alpha-transducin (GNAT1)(NM_(—)000172);

(j) SEQ ID NO: 24 guanylate cyclase activator 1A (GUCA1A)(NP_(—)000400.2);

(k) SEQ ID NO: 25 retina specific guanylate cyclase (GUCY2D),(NP_(—)000171.1);

(l) SEQ ID NO: 26 & 27 alpha subunit of the cone cyclic nucleotide gatedcation channel (CNGA3) (NP_(—)001073347.1 or NP_(—)001289.1);

(m) SEQ ID NO: 28 Human cone transducin alpha subunit (incompleteachromotopsia);

(n) SEQ ID NO: 29 cone cGMP-specific 3′,5′-cyclic phosphodiesterasesubunit alpha′, protein (cone dystrophy type 4);

-   -   (o) SEQ ID NO: 30 retinal cone rhodopsin-sensitive cGMP        3′,5′-cyclic phosphodiesterase subunit gamma, protein (retinal        cone dystrophy type 3A);

(p) SEQ ID NO: 31 cone rod homeobox, protein (Cone-rod dystrophy);

(q) SEQ ID NO: 32 cone photoreceptor cyclic nucleotide-gated channelbeta subunit, protein (achromatopsia);

(r) SEQ ID NO: 33 cone photoreceptor cGMP-gated cation channelbeta-subunit, protein (total color blindness, for example, amongPingelapese Islanders);

(s) SEQ ID NO: 35 (SEQ ID NO: 34) retinitis pigmentosa 1 (autosomaldominant) (RP 1);

(t) SEQ ID NO: 37 (SEQ ID NO: 36) retinitis pigmentosa GTPase regulatorinteracting protein 1 (RPGRIP 1);

(u) SEQ ID NO: 39 (SEQ ID NO: 38) PRP8;

(v) SEQ ID NO: 41 (SEQ ID NO: 40) centrosomal protein 290 kDa (CEP290);

(w) SEQ ID NO: 43 (SEQ ID NO: 42) IMP (inosine 5′-monophosphate)dehydrogenase 1 (IMPDH1), transcript variant 1;

(x) SEQ ID NO: 45 (SEQ ID NO: 44) aryl hydrocarbon receptor interactingprotein-like 1 (AIPL1), transcript variant 1;

(y) SEQ ID NO: 47 (SEQ ID NO: 46) retinol dehydrogenase 12(all-trans/9-cis/11-cis) (RDH12);

(z) SEQ ID NO: 49 (SEQ ID NO: 48) Leber congenital amaurosis 5 (LCA5),transcript variant 1; and

(aa) exemplary OPN1LW/OPN1MW2 polymorphs (compared to OPN1LW (L opsin)polypeptide sequence; the amino acid to the left of the number is theresidue present in the L opsin sequence; the number is the reside numberin L opsin, and the reside to the right of the number is the variationfrom L opsin. Polymorphs according to these embodiments may comprise oneor more of the amino acid substitutions in Table 1 below:

TABLE 1 (i) Thr65Ile (ii) Ile111Val (iii) Ser116Tyr (iv) Leu153Met (v)Ile171Val (vi) Ala174Val (vii) Ile178Val (viii) Ser180Ala (ix) Ile230Thr(x) Ala233Ser (xi) Val236Met (xii) Ile274Val (xiii) Phe275Leu (xiv)Tyr277Phe (xv) Val279Phe (xvi) Thr285Ala (xvii) Pro298Ala (xviii)Tyr309Phe.

The proteins recited in (a)-(c) and (aa) are all involved in colorvision. The exemplary polymorphs include ones at positions 65, 116, 180,230, 233, 277, 285, and 309 that affect the spectra of the pigments incone cells expressing them. Positions 274, 275, 277, 279, 285, 298 and309 together distinguish L opsin from M opsin.

The proteins recited (d)-(z) are exemplary eye disease-associated gene,such as in retinitis pigmentosa (polypeptides “e”-“l”, “s”-“y”),incomplete achromatopsia (polypeptide “m”), Stargardt's (polypeptide“d”); Leber congenital amaurosis (polypeptide “z”); cone dystrophy, suchas cone dystrophy type 4 (polypeptide “n”); retinal cone dystrophy ; forexample, retinal cone dystrophy type 3A (polypeptide “o”) ; Cone-roddystrophy (polypeptide “p”); achromatopsia (polypeptide “q′); and totalcolor blindness, for example, among Pingelapese Islanders (polypeptide“r”).

Exemplary nucleic acids encoding these polypeptides are shown by SEQ IDNO in parenthesis. Thus, in a further preferred embodiment, the genescomprise or consist of a nucleic acid sequence according to one or moreof the nucleic acid sequences recited above. In a further preferredembodiment, the vector comprises the sequence shown in SEQ ID NO: 50

Any suitable gene therapy vector that can be used for cone cell deliverycan be used in the methods of the present invention; the vector maycomprise single or double stranded nucleic acid; preferably singlestranded or double stranded DNA. In a preferred embodiment that can becombined with any of the above embodiments, the gene delivery vectorcomprises a recombinant adeno-associated virus (AAV) gene deliveryvector. Prior to the present invention, rAAV vectors had not been showncapable of transducing primate cone cells. In this embodiment, the genedelivery vector is bounded on the 5′ and 3′ end by functional AAVinverted terminal repeat (ITR) sequences. By “functional AAV ITRsequences” is meant that the ITR sequences function as intended for therescue, replication and packaging of the AAV virion. Hence, AAV ITRs foruse in the vectors of the invention need not have a wild-type nucleotidesequence, and may be altered by the insertion, deletion or substitutionof nucleotides or the AAV ITRs may be derived from any of several AAVserotypes. The rAAV vector may be derived from an adeno-associated virusserotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4,AAV-5, AAV-6, AAV-7, AAV-8, etc. Preferred AAV vectors have the wildtype REP and CAP genes deleted in whole or part, but retain functionalflanking ITR sequences. In a further preferred embodiment, the AAVvector comprises rAAV2/5, a “pseudotyped” version of AAV2 created byusing rep from AAV2 and cap from A AV5 or AAV2, A AV3, AAV4, AAV6, AAV7,AA V8 together with a plasmid containing a vector based on AAV2.Preferably, the rAAV is replication defective, in that the AAV vectorcannot independently further replicate and package its genome. Forexample, when cone cells are transduced with rAAV virions, the gene isexpressed in the transduced cone cells, however, due to the fact thatthe transduced cone cells lack AAV rep and cap genes and accessoryfunction genes, the rAAV is not able to replicate.

Recombinant AAV (rAAV) virions encapsulating the vectors recited abovefor use in transducing cone cells may be produced using standardmethodology. In one embodiment, an AAV expression vector according tothe invention is introduced into a producer cell, followed byintroduction of an AAV helper construct, where the helper constructincludes AAV coding regions capable of being expressed in the producercell and which complement AAV helper functions absent in the AAV vector.This is followed by introduction of helper virus and/or additionalvectors into the producer cell, wherein the helper virus and/oradditional vectors provide accessory functions capable of supportingefficient rAAV virus production. The producer cells are then cultured toproduce rAAV. These steps are carried out using standard methodology.Replication-defective AAV virions encapsulating the recombinant AAVvectors of the instant invention are made by standard techniques knownin the art using AAV packaging cells and packaging technology. Examplesof these methods may be found, for example, in U.S. Pat. Nos. 5,436,146;5,753,500, 6,040,183, 6,093,570 and 6,548,286, expressly incorporated byreference herein in their entirety. Further compositions and methods forpackaging are described in Wang et al. (US 2002/0168342), alsoincorporated by reference herein in its entirety.

Any suitable method for producing viral particles for delivery can beused, including but not limited to those described in the examples thatfollow. Any concentration of viral particles suitable to effectivelytransducer cone cells can be administered to the eye. In one preferredembodiment, viral particles are delivered in a concentration of at least10¹⁰ vector genome containing particles per mL; in various preferredembodiments, the viral particles are delivered in a concentration of atleast 7.5×10¹⁰; 10¹¹; 5×10¹¹; 10¹²; 5×10¹²; 10¹³; 1.5×10¹³; 3×10¹³;5×10¹³; 7.5×9×10¹³; or 9×10¹³ vectorgenome containing particles per mL.Similarly, any total number of viral particles suitable to provideappropriate transduction of retinal cone cells can be administered tothe primate's eye. In various preferred embodiments, at least 10¹⁰;5×10¹⁰; 10¹¹; 5×10¹¹; 10¹²; 1.5×10¹²; 3×10¹²; 5×10¹²; 7.5×10¹²; 10¹³;1.5×10¹³; or 2.7×10¹³ viral particles are injected per eye. Any suitablenumber of administrations of the vector to the primate eye can be made.In one embodiment, the methods comprise a single administration; inother embodiments, multiple administrations are made over time as deemedappropriate by an attending clinician.

The viral stock for delivery to the primate eye can be treated asappropriate for delivery. The viral stock can be combined withpharmaceutically-acceptable carriers, diluents and reagents useful inpreparing a formulation that is generally safe, non-toxic, anddesirable, and includes excipients that are acceptable for primate use.Such excipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous. Examples of such carriers or diluentsinclude, but are not limited to, water, saline, Ringer's solutions,dextrose solution, and 5% human serum albumin. Supplementary activecompounds can also be incorporated into the formulations. Solutions orsuspensions used for the formulations can include a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial compounds such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelatingcompounds such as ethylenediaminetetraacetic acid (EDTA); buffers suchas acetates, citrates or phosphates; detergents such as Tween 20 toprevent aggregation; and compounds for the adjustment of tonicity suchas sodium chloride or dextrose. The pH can be adjusted with acids orbases, such as hydrochloric acid or sodium hydroxide.

In a further preferred embodiment that can be combined with any otherembodiment, the methods of the invention restore visual capacity in theprimate. As used herein, “restoring visual capacity” means that somebenefit to vision is provided, including but not limited to a reductionor slowing of vision loss; improved visual acuity; decrease in abnormalsensitivity to bright lights; and/or an increase in one or more visualattributes, such as improved color perception (ie: monochromatic todichromatic vision; dichromatic to trichromatic vision; trichromatic totetrachromatic vision; etc.). The primate is preferably of the ParvorderCatarrhini, and more preferably is a human.

In a further preferred embodiment that can be combined with all of theabove embodiments, the primate suffers from color blindness, and theprimate is able to visualize new colors as a result of the therapy. Inthis embodiment, it is preferred that the enhancer (if present)comprises or consists of a sequence selected from the group consistingof the L/M minimal opsin enhancer (SEQ ID NO: 51), L/M enhancer elementsof 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, or more nucleotides thatcomprise one or more copies of the L/M minimal opsin enhancer, and thefull L/M opsin enhancer (SEQ ID NO:4), or other portions thereofsuitable to promote expression in a cone-specific manner, and thepromoter comprises or consists of a sequence selected from the groupconsisting of L opsin promoter (SEQ ID NO: 1), the M opsin promoter (SEQID NO:2), and the S opsin promoter (SEQ ID NO:3), while the gene encodesone or more polypeptides comprising or consisting of a sequence selectedfrom the group consisting of SEQ ID NO: 7 (OPN1SW), SEQ ID NO: 9(OPN1MW), SEQ ID NO: 11 (OPN1LW), mRNA; and exemplary OPN1LW/OPN1MW2polymorphs as described in Table 1 above. It is further preferred thatthe vector comprises a rAAV vector as described above.

The color blindness may be acquired or inherited, and can be full(monochromatic) or partial. In a preferred embodiment, the primate haspartial color blindness selected from the group consisting of red-greenand blue-yellow color blindness. The partial color blindness cancomprise, for example, dichromacy or anomalous trichromasy. Thesemethods result in the primate improved color perception (ie:monochromatic to dichromatic vision; dichromatic to trichromatic vision;trichromatic to tetrachromatic vision; etc.). The primate is preferablyof the Parvorder Catarrhini, and more preferably is a human.

As described in detail below, the methods may be used to improve colorperception in primates from dichromatic to trichromatic. Dichromats whoare missing either the L- or the M-photopigment fail to distinguish fromgrey:colours near the so-called ‘spectral neutral point’ located in thebluegreen region of color space (near dominant wavelength of 490 nm) andcomplementary colors near the ‘extra-spectral neutral point’ in thered-violet region (near dominant wavelength of 499 nm), Co-expressingthe L-opsin transgene within a subset of endogenous M-cones shiftedtheir spectral sensitivity to respond to long wavelength light, thusproducing two distinct cone types absorbing in the middle-to-longwavelengths, as required for trichromasy. These results demonstrate thatgene therapy changed the spectral sensitivity of a subset of the cones,and the results further demonstrate the unexpected result that adultmonkeys gained new color vision capacities because of the gene therapy.

In a further preferred embodiment of all of the above embodiments, theprimate has a vision disorder in which its photoreceptors are healthy,such as color blindness. As used herein, “healthy” means that the cellsbeing treated are functioning but simply do not provide for the desiredcolor perception, in contrast to gene therapy in which the target cellsare degenerating or dying. The studies reported herein are the first touse gene therapy in primates to address a vision disorder in which allphotoreceptors are intact and healthy, making it possible to assess thefull potential of gene therapy to restore visual capacities. The methodsof the present invention thus will allow many opportunities forfunctions to be added or restored in the eye.

In a further preferred embodiment that can be combined with any of theother embodiments herein, the primate is an adult primate, such as anadult human (ie: at least 16 years of age; preferably at least 18 yearsof age or 21 years of age). Classic visual deprivation experiments haveled to the expectation that neural connections established duringdevelopment would not appropriately process an input that was notpresent from birth. Therefore, it was believed that treatment ofcongenital vision disorders would be ineffective unless administered tothe very young. The present study thus provides significantly unexpectedresults in curing a visual disorder in an adult primate.

Those of skill in the art will readily appreciate, based on theteachings herein, the variety of treatment modalities that can beaccomplished using the methods of the invention. In one embodiment, thegene encodes ABCR and is administered to the eye of a primate withStargardt disease. In other embodiments, the gene encodes:

one or more of polypeptides “e”- “1” and “s”- “y” in Table 1, and isadministered to the eye of a primate with retinitis pigmentosa;

polypeptide “m” in Table 1, and is administered to the eye of a primatewith incomplete achromatopsia;

polypeptide “z” in Table 1, and is administered to the eye of a primatewith Leber congenital amaurosis;

polypeptide “n” in Table 1, and is administered to the eye of a primatewith cone dystrophy, such as cone dystrophy type 4;

polypeptide “o” in Table 1, and is administered to the eye of a primatewith retinal cone dystrophy, for example, retinal cone dystrophy type3A;

polypeptide “p” in Table 1, and is administered to the eye of a primatewith cone-rod dystrophy;

polypeptide “q” in Table 1, and is administered to the eye of a primatewith achromatopsia; and/or

polypeptide “r” in Table 1, and is administered to the eye of a primatewith total color blindness, for example, a native of the PingelapeseIslands.

Any suitable means for delivery of the gene therapy vector to the eyecan be used, including but not limited to administering in a contactlens fluid, contact lens cleaning and rinsing solutions, eye drops,surgical irrigation solutions, ophthalmological devices, injection,iontophoresis, topical instillation on the eye, and topicalinstillation. The topical instillation can be administered, for example,in the form of a liquid solution, a paste, of a hydrogel. The topicalinstillation can be embedded, for example, in a foam matrix or supportedin a reservoir. The injection into the primate eye can be, for example,an intracameral injection, an intracorneal injection, a subconjonctivalinjection, a subtenon injection, a subretinal injection, an intravitrealinjection, and an injection into the anterior chamber.

The primate's progress in response to the treatment may be monitored byany suitable means. In embodiments where the methods are used to treatcolor blindness, monitoring or progress may comprise, for example, useof standard color vision tests, or the wide-field color multifocalelectroretinogram (mf-ERG) system described below to detect spectralsensitivity shifts in the primate's vision. Thus, in another aspect, thepresent invention provides methods for use of the electroretinogramdisclosed herein for monitoring changes in vision perception, such ascolor perception, of a primate undergoing gene therapy to treat a visualdisorder. All embodiments of the methods disclosed above can be combinedwith all embodiments of the electroretinogram disclosed below.

In a second aspect, the present invention provides isolated nucleic acidexpression vectors comprising:

(a) a promoter region, wherein the promoter region is specific forprimate retinal cone cells; and

(b) a gene encoding a therapeutic, wherein the gene is operativelylinked to the promoter region.

All terms in this second aspect have the same meaning as disclosed abovefor the first aspect of the invention. Similarly, all embodiments andcombinations thereof disclosed above in the first aspect of theinvention can be used in this second aspect of the invention. Theinventors have demonstrated effective treatment of congenital visiondisorders in adult primates using the recombinant vectors of theinvention, a result that is completely unexpected in the art.

Any suitable promoter region can be used in the isolated nucleic acidexpression vector, so long as it specifically promotes expression of thegene in retinal cone cells. In a preferred embodiment, the promoterspecifically promotes expression of the gene in Catarrhini retinal conecells; even more preferably in human retinal cone cells. Exemplarysuitable promoter regions include the promoter region for anycone-specific gene, such as the L opsin promoter (SEQ ID NO: 1), the Mopsin promoter (SEQ ID NO: 2), and the S opsin promoter (SEQ ID NO: 3),or portions thereof suitable to promote expression in a cone-specificmanner.

In a preferred embodiment, the isolated nucleic acid expression vectorfurther comprises an enhancer element upstream of the promoter, whereinthe gene is operatively linked to the enhancer element. Any suitableenhancer element can be used in the gene therapy vectors, so long as itenhances expression of the gene when used in combination with thepromoter. In a preferred embodiment, the enhancer element is specificfor retinal cone cells; more preferably, it is specific for primateretinal cone cells; more preferably in Catarrhini retinal cone cells;even more preferably in human retinal cone cells. Exemplary suitableenhancer regions comprise or consist of the enhancer region for anycone-specific gene, such as the L/M minimal opsin enhancer (SEQ ID NO:51), L/M enhancer elements of 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,or more nucleotides that comprise one or more copies of the L/M minimalopsin enhancer, and the full L/M opsin enhancer (SEQ ID NO: 4), or otherportions thereof suitable to promote expression in a cone-specificmanner.

The length of the promoter and enhancer regions can be of any suitablelength for their intended purpose, and the spacing between the promoterand enhancer regions can be any suitable spacing to promotecone-specific expression of the gene product. In various preferredembodiments, the enhancer is located 0-1500; 0-1250; 0-1000; 0-750;0-600; 0-500; 0-400; 0-300; 0-200; 0-100; 0-90; 0-80; 0-70; 0-60; 0-50;0-40; 0-30; 0-20; or 0-10 nucleotides upstream of the promoter. Thepromoter can be any suitable distance upstream of the encoded gene.

In a further preferred embodiment that can be combined with any otherembodiment in any aspect of the present invention, the enhancercomprises or consists of a sequence selected from the group consistingof the L/M minimal opsin enhancer (SEQ ID NO: 51), L/M enhancer elementsof 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, or more nucleotides thatcomprise one or more copies of the L/M minimal opsin enhancer, and thefull L/M opsin enhancer (SEQ ID NO: 4), or other portions thereofsuitable to promote expression in a cone-specific manner, and thepromoter comprises or consists of a sequence selected from the groupconsisting of L opsin promoter (SEQ ID NO: 1), the M opsin promoter (SEQID NO: 2), and the S opsin promoter (SEQ ID NO: 3).

In a further preferred embodiment, the isolated nucleic acid expressionvector further comprises an intron comprising a splice donor/acceptorregion, wherein the intron is located downstream of the promoter regionand is located upstream of the gene. Any intron can be used, so long asit comprises a splice donor/acceptor region recognized in primate conecells, so that the intron can be spliced out of the resulting mRNAproduct. In one embodiment, the intron comprises or consists of an SV40intron according to SEQ ID NO: 5. In various preferred embodiments, the3′ end of the intron is 0-20; 0-15; 0-10; 0-9; 0-8; 0-7; 0-6; or 0-5nucleotides upstream of the gene, and its 5′ end is 0-20; 0-15; 0-10;0-9; 0-8; 0-7; 0-6; or 0-5 nucleotides downstream of the proximalpromoter region.

The gene is operatively linked to the promoter region and the enhancerelement, such that the promoter and enhancer elements are capable ofdriving expression of the gene or cDNA in cone cells of the subject.

The gene encoding a therapeutic to be expressed in the cone cells can beany gene or cDNA that encodes a polypeptide or RNA-based therapeutic(siRNA, antisense, ribozyme, shRNA, etc.) that can be used as atherapeutic for treating a cone cell disorder, or as a means tootherwise enhance vision, including but not limited to promotingtetrachromatic color vision. In various preferred embodiments, the geneencodes a therapeutic protein comprising or consisting of thosedisclosed above in the methods of the first aspect of the invention.

In a further preferred embodiment, the vector comprises the sequenceshown in SEQ ID NO: 50, which details the vector used in at least someof the examples that follow.

In a further preferred embodiment that can be combined with any of theabove embodiments, the gene delivery vector comprises a recombinantadeno-associated virus (AAV) gene delivery vector. In a furtherpreferred embodiment, the AAV vector comprises rAAV2/5. Preferably, therAAV is replication defective, in that the AAV vector cannotindependently further replicate and package its genome. For example,when cone cells are transduced with rAAV virions, the gene is expressedin the transduced cone cells, however, due to the fact that thetransduced cone cells lack AAV rep and cap genes and accessory functiongenes, the rAAV is not able to replicate.

In a third aspect, the present invention provides a formulationcomprising packaged viral particles containing the nucleic acidexpression vectors of the second aspect of the invention. In onepreferred embodiment, viral particles are present in a concentration ofat least 10¹⁰ vector genome containing particles per mL; in variouspreferred embodiments, the viral particles are delivered in aconcentration of at least 7.5×10¹⁰; 10¹¹; 5×10¹¹; 10¹²; 5×10¹²; 10¹³;1.5×10¹³; 3×10¹³; 5×10¹³; 7.5×9×10¹³; or 9×10¹³ vectorgenome containingparticles per mL. The formulation may further comprisepharmaceutically-acceptable carriers, diluents and reagents as describedabove in the first aspect of the invention. The formulation may be inthe form of a liquid solution, a paste, a hydrogel, or may be embeddedwithin a substrate, including but not limited to a foam matrix orsupported in a reservoir.

In a fourth aspect, the present invention provides recombinant hostcells transfected or transduced with the nucleic acid expression vectorof the second aspect of the invention. The cells may be of any type thatcan be transfected with the expression vector. In one embodiment wherethe expression vector is a rAAV vector, the cells comprise producercells transduced with a replication incompetent rAAV expression vectoraccording to the second aspect of the invention, form which viralparticles can be obtained by introduction of an AAV helper construct asdescribed above and as is well known in the art.

In a fifth aspect, the present invention provides a color multifocalelectroretinogram system, comprising:

(a) an electroretinogram (ERG) comprising

-   -   (i) a recording electrode that is (A) designed for placement on        at least one of a cornea and a sclera of at least one eye of a        subject and (B) arranged to output at least one signal generated        by the at least one eye; and    -   (ii) a computing system communicatively coupled to the recording        electrode, the computing system comprising (A) at least one        processor and    -   (B) data storage containing instructions executable by the at        least one processor to carry out a set of functions, the set of        functions including processing and saving the at least one        signal generated by the at least one eye;

(b) a retinal stimulator comprising matched light sources selected fromthe group consisting of red, green, blue, and ultraviolet light sources,wherein the matched light sources are connected to the ERG and inoperation can be independently frequency modulated at rates betweenabout 1 Hz and about 60 Hz, inclusive, wherein the stimulator inoperation is capable of stimulating a retinal field of a subjectthroughout an operating radius of at least about 70 degrees;

(c) one or more constant current integrated circuit chips arranged todrive the stimulator; and

(d) a pulse-frequency modulator connected to the retinal stimulator,wherein in operation the pulse-frequency modulator is capable ofcontrolling individual stimulator segments while keeping relativespectral content of the light constant.

The electroretinograms of the present invention can be used, forexample, in characterizing the topography of expression of the differentopsin transgenes in the eyes of living subjects treated with genetherapy, and thus can be used with the gene therapy methods of theinvention disclosed above.

As used here, a “matched” light source is one that includes lightstimulus of different wavelengths, wherein the number of pixels isapproximately the same, or is the same, at each wavelength. Onenon-limiting example is a matched light source stimulus containing 1024doublet pixels each containing a red (653 nm, half-bandwidth 22 nm) anda green (527 nm, half-bandwidth 33 nm) LED, with a resulting matchedlight source with 2,048 paired green and red LEDs.

In various preferred embodiments, the stimulator in operation is capableof stimulating a retinal field of a subject throughout an operatingradius of at least about 80, 90, 100, 110, 120, 130, 140, 150, or moredegrees.

In one preferred embodiment, the matched light sources are paired redand green light sources. In another preferred embodiment, the matchedlight sources are triplets of red, green, and blue light sources. In afurther preferred embodiment, the matched light sources are quartets ofred, green, blue, and ultraviolet light sources.

Any suitable matched light source can be used. In one preferredembodiment, the matched light sources comprise matched light emittingdiodes (LEDs).

In another preferred embodiment, that can be combined with any of theembodiments herein, the retinal stimulator comprises a concave surfacecomprising a series of trapezoidal-shaped circuit boards placededge-to-edge, wherein the concave surface positions the matched lightsources so in operation they are held equidistantly from and pointingtoward a single focal point where a subject's pupil can be positioned.This embodiment helps to limit SNR fall-off in peripheral retinalregions. Any suitable concave surface can be used; in a preferredembodiment, the concave surface comprises a geodesic dome.

In a further embodiment that can be combined with any of the aboveembodiments, the set of functions executable by the processor furthercomprises coding and decoding topographical regions on the recordingelectrode using a cyclic summation technique.

In a further preferred embodiment that can be combined with any of theembodiments herein, the ERG further comprises an amplifier; and whereinthe computing system is communicatively coupled to the amplifier.

Further embodiments and details of the color multifocalelectroretinogram system are provided in the Examples that follow.

In a further aspect, the present invention provides methods fordetermining a location of functioning opsin expression in a subject,comprising use of the mf-ERG of any embodiment or combination ofembodiments of the fifth aspect of the invention, wherein the recordingelectrode is placed on at least one of a cornea and a sclera of at leastone eye of a subject; stimulating the subject's retinal field with theretinal stimulator; and determining responses of different areas of thesubject's retina to different stimulation frequencies to generate a mapof retinal responses, wherein the map provides a location of functioningopsin expression in a subject. In one preferred embodiment, the subjecthas been treated according to the gene therapy methods for colorblindness disclosed above according to any embodiment or combination ofembodiments of the first aspect of the invention.

Unless the context clearly dictates otherwise, embodiments in one aspectof the invention may be used in other aspects of the invention, and canbe combined with each other.

EXAMPLE 1

Red-green colour blindness, which results from the absence of either thelong-(L) or middle-(M) wavelength-sensitive visual photopigments, is themost common single locus genetic disorder. Here, the possibility ofcuring colour blindness using gene therapy was explored in experimentson adult monkeys that had been colour blind since birth. A third type ofcone pigment was added to dichromatic retinas, providing the receptoralbasis for trichromatic colour vision. This opened a new avenue toexplore the requirements for establishing the neural circuits for a newdimension of colour sensation. Classic visual deprivation experiments'have led to the expectation that neural connections established duringdevelopment would not appropriately process an input that was notpresent from birth. Therefore, it was believed that treatment ofcongenital vision disorders would be ineffective unless administered tothe very young. Here, however, addition of a third opsin in adultred-green colour-deficient primates was sufficient to producetrichromatic colour vision behaviour. Thus, trichromacy can arise from asingle addition of a third cone class and it does not require an earlydevelopmental process. This provides a positive outlook for thepotential of gene therapy to cure adult vision disorders.

Gene therapy was performed on adult squirrel monkeys (Saimiri sciureus)that were missing the L opsin gene. In this species, some females havetrichromatic colour vision while males are red-green colour blind².Serotype 2/5 recombinant adeno-associated virus (rAAV) containing ahuman L-opsin gene under control of the L/M opsin enhancer and promoter(FIG. 1 a) was delivered to the photoreceptor layer via subretinalinjections. Transcriptional regulatory elements were chosen to directexpression preferentially in M cones, but not short-(S)wavelength-sensitive cones or rods³. To provide the receptoral basis fortrichromacy, animals received three 100 μL injections (containing atotal of 2.7×10¹³ viral particles) in each eye which produced arelatively uniform, third submosaic of approximately 15-36% of M conesthat coexpressed the transgene (FIGS. 1 e, f).

Prior to treatment, monkeys were trained to perform a computer-basedcolour vision test, the Cambridge Colour Test^(4,5), which was modifiedfor use with animals⁶ (FIG. 2 a). Dichromats who are missing either theL- or M-photopigment fail to distinguish from grey: colours near theso-called “spectral neutral point” located in the blue-green region ofcolour space (near dominant wavelength (DW) 490 nm) and complementarycolours near the “extra-spectral neutral point,” in the red-violetregion (near DW=−499 nm). While trichromats have four main huepercepts—blue, yellow, red, and green—dichromats have only two percepts,nominally blue and yellow. Before treatment, two dichromatic monkeyscompleted three colour vision tests consisting of 16 hues (FIG. 2 b, c).Four-to-six months was required to test all 16 hues; thus, baselineresults represent testing conducted for more than a year. As predicted,prior to treatment monkeys had low thresholds (averaging <0.03 units inu′, v′ colour space) for colours that represent blues and yellows totheir eyes, but always failed to discriminate the blue-green (DW=490 nm)and red-violet hues (DW=−499 nm) with thresholds extrapolated frompsychometric functions being orders of magnitude higher (FIGS. 2 b, c).Results were highly repeatable, with no improvement between the firstand third tests, making us confident that animals would notspontaneously improve in the absence of treatment. Co-expressing theL-opsin transgene within a subset of endogenous M-cones shifted theirspectral sensitivity to respond to long wavelength light, thus producingtwo distinct cone types absorbing in the middle-to-long wavelengths, asrequired for trichromacy. The spectral sensitivity shift was readilydetected using a custom-built wide-field colour multifocalelectroretinogram (mf-ERG) system (FIGS. 1 b, c, f) (see ref 7 fordetails). In preliminary experiments, validity of the colour mf-ERG wastested using an animal that had received a mixture of the L-opsin-codingvirus plus an identical virus, except that a green fluorescent protein(GFP) gene replaced the L-opsin gene. As reported previously, faint GFPfluorescence was first detected at 9 weeks post-injection, and itcontinued to increase in area and intensity through 24 weeks⁸. Whilefaint signs of GFP were first detectable at 9 weeks, L-opsin levelssufficient to produce suprathreshold mf-ERG signals were still notpresent at 16 weeks post-injection (FIG. 1 c, inset). After GFPfluorescence became robust, the red light mf-ERG, which indicatesresponses from the introduced L-opsin, showed highly elevated responseamplitudes in two areas (FIG. 1 c) corresponding to locations ofsubretinal injections (FIG. 1 d).

The two dichromatic monkeys who participated in behavioural tests ofcolour vision were treated with only L-opsin-coding virus. While theelongated pattern produced by two injections in FIGS. 1 c and d allowedmf-ERG validation, the treatment goal was to produce a homogeneousregion, as resulted from 3 injections shown in f, where the highestmf-ERG response covered about 80° of central retina, roughly the areafor which humans have good red-green discrimination. These resultsdemonstrate that gene therapy changed the spectral sensitivity of asubset of the cones. A priori, there were two possibilities for how achange in spectral sensitivity might change colour vision behaviour: 1)animals may have an increase in sensitivity to long-wavelength light,but if the neural circuitry for extracting colour information from thenascent “M+L cone” submosaic was absent, they would remain dichromatic,the hallmark of which is having two hues that are indistinguishable fromgrey (FIG. 2 d). The spectral neutral point for individuals that haveonly S- and M-cones, (e.g. monkeys 1 and 2 pre-therapy), occurs neardominant wavelength (DW)=495 nm. At the limit, an increase in spectralsensitivity would shift the monkeys' neutral point toward that ofindividuals with only S and L cones, near DW=505 nm (dashed blue lines,FIG. 2 d). 2) The second, more engaging possibility was that treatmentwould be sufficient to expand sensory capacity in monkeys, providingthem with trichromatic vision. In this case, the animals' post-therapyresults would appear similar to FIG. 2 e, obtained from a trichromaticfemale control monkey.

Daily testing continued after treatment. After about 20 weekspost-injection (arrow, FIG. 3 a), the trained monkeys' thresholds forblue-green and red-violet (DWs=490 and −499 nm, respectively, FIGS. 3 b,c) improved, reducing to an average of 0.08 units in u′, v′ colourspace, indicating that they gained trichromatic vision. This time pointcorresponded to the same period in which robust levels of transgeneexpression were reported in the squirrel monkey⁸. A trichromatic femalemonkey and untreated dichromatic monkeys were tested in parallel. Asexpected, the female had low thresholds for all colours, averaging <0.03units in u′, v′ colour space, but the untreated dichromats always failedto discriminate DWs=490 nm (triangle, FIGS. 3 a) and −499 nm, indicatinga clear difference between treated and untreated monkeys.

Early experiments in which we obtained negative results served as “shamcontrols,” demonstrating that acquiring a new dimension of colour visionrequires a shift in spectral sensitivity that results from expression ofan L pigment in a subset of M cones. Using similar subretinal injectionprocedures, we delivered fewer viral particles of an L-opsin-codingrAAV2/5 virus with an extra 146 base pair (bp) segment near the splicedonor/acceptor site that had been carried over from the cloning vectorand that was absent in the GFP-coding rAAV2/5 virus. The 146 bp segmentcontained an ATG and a duplicate mRNA start site that may haveinterfered with expression (see Full Methods online). Three monkeysreceived injections of this vector, containing an average of 1.7×10¹²virus particles per eye, and no reliable changes in spectral sensitivitywere measured using the ERG. One animal was also tested behaviourallyand his colour vision was unchanged from baseline 1 year afterinjection. In subsequent experiments reported here, we removed the extra146 bp segment and also increased the amount of viral particlesdelivered per eye by approximately 16-fold, to 2.7×10¹³. Negativeresults from earlier injections demonstrated that the subretinalinjection procedure itself does not produce changes in the ERG or incolour vision.

The change in spectral sensitivity measured with the mf-ERG is necessarybut not sufficient to produce a new colour vision capacity. For example,individuals with L but no M cones (termed deuteranopes) have arelatively enhanced sensitivity to red light but they are still asdichromatic as individuals with M but no L cones (protanopes), in thatthey are unable to distinguish particular “colours” from grey. To verifythat the behavioural change observed in animals expressing the L pigmenttransgene was not purely a shift in spectral sensitivity (see FIG. 2 d),monkey 1 was also tested on DWs=496 and 500 nm, and monkey 2 was testedon DWs 496 and 507 nm. Together, these DWs span the possible confusionpoints for deuteranopes and protanopes and for any intermediatedichromatic forms that could arise from expressing combinations of L andM pigments. As shown in FIGS. 3 b and c, both monkeys' measuredthresholds for these additional hues were similar to their thresholdsfor DW=490 nm, demonstrating they now lacked a spectral neutral pointand have become truly trichromatic. Furthermore, treated monkeys wereable to discriminate blue-green (DW=490 nm) when it was tested against ared-violet background (DW=−499 nm), instead of the grey background,indicating that the monkeys' newly-acquired “green” and “red” perceptswere distinct from one another. The treated monkeys' improvement incolour vision has remained stable for over 2 years and we plan tocontinue testing the animals to evaluate long term treatment effects.

Classic experiments in which visual deprivation of one eye duringdevelopment caused permanent vision loss¹ led to the idea that inputsmust be present during development for the formation of circuits toprocess them. From the clear change in behaviour associated withtreatment, compared both between and within subjects, we conclude thatadult monkeys gained new colour vision capacities because of genetherapy. These startling empirical results provide insight into theevolutionary question of what changes in the visual system are requiredfor adding a new dimension of colour vision. Previously, it seemedpossible that a transformation from dichromacy to trichromacy wouldrequire evolutionary/developmental changes, in addition to acquiring athird cone type. For example, L and M opsin-specific genetic regulatoryelements might have been required to direct the opsins into distinctcone types⁹ that would be recognized by L and M cone-specific retinalcircuitry¹⁰, and to account for cortical processing, multi-stagecircuitry” might have evolved specifically for the purpose oftrichromacy. However, our results demonstrate that trichromatic colourvision behaviour requires nothing more than a third cone type. As analternative to the idea that the new dimension of colour vision arose byacquisition of a new L vs. M pathway, it is possible that it exploitedthe pre-existing blue-yellow circuitry. For example, if addition of thethird cone class split the formerly S vs. M receptive fields into twotypes with differing spectral sensitivities, this would obviate the needfor neural rewiring as part of the process of adopting new colourvision.

Some form of inherent plasticity in the mammalian visual system can beinferred from the acquisition of novel colour vision, as was alsodemonstrated in genetically engineered mice¹²; however, the point hasbeen made that such plasticity need not imply that any rewiring of theneural circuitry has occurred¹³. Similarly, given the fact that newcolour vision behaviour in adult squirrel monkeys corresponded to thesame time interval as the appearance of robust levels of transgeneexpression, we conclude that rewiring of the visual system was notassociated with the change from dichromatic to trichromatic vision.

Treated adult monkeys unquestionably respond to colours previouslyinvisible to them. The internal experiences associated with the dramaticchange in discrimination thresholds measured here cannot be determined;therefore, we cannot know whether the animals experience new internalsensations of “red” and “green.” Nonetheless, we do know that evolutionacts on behaviour, not on internalized experiences, and we suggest thatgene therapy recapitulated what occurred during evolution of trichromasyin primates. These experiments demonstrate that a new colour visioncapacity, as defined by new discrimination abilities, can be added bytaking advantage of pre-existing neural circuitry and, internalexperience aside, full colour vision could have evolved in the absenceof any other change in the visual system except the addition of a thirdcone type.

Gene therapy trials are underway for Leber's congenital amaurosis¹⁴⁻¹⁶.Thus far, treatment has been administered to individuals who havesuffered retinal degeneration from the disease. The experiments reportedhere are the first to use gene therapy in primates to address a visiondisorder in which all photoreceptors are intact and healthy, making itpossible to assess the full potential of gene therapy to restore visualcapacities. Treatment allowing monkeys to see new colours in adulthoodprovides a striking counter-example to what occurs under conditions ofmonocular deprivation. For instance, it is impossible to restore visionin an adult who had grown up with a unilateral cataract. Futuretechnologies will allow many opportunities for functions to be added orrestored in the eye. While some changes may produce outcomes analogousto monocular deprivation, we predict that others, like gene therapy forred-green colour blindness, will provide vision where there waspreviously blindness.

METHODS SUMMARY FOR EXAMPLE 1

Viral vector. CHOPS2053 was a 2.1 kb fragment containing the locuscontrol region (LCR) and proximal promoter (PP) upstream of the humanX-chromosome opsin gene array^(9,17). These elements (also known aspR2.1) have been shown to target transgene expression to mammalian L/Mcones^(3,18). RHLOPS was a 1.2 kb fragment containing recombinant humanL opsin cDNA. A clone of the human L opsin cDNA¹⁹, known as hs7, wasgenerously provided by J. Nathans. The QuickChange kit (Stratagene) wasused to convert codon 180 so that it would encode a human L pigmentmaximally sensitive to 562 nm²⁰. The virus was made using the genomefrom rAAV serotype 2 and the capsid from serotype 5, and the preparationhad 9×10¹³ DNase-resistant vector genome containing particles per mL. Toprevent vector aggregation, 0.014% Tween 20 was added to the finalvector preparation. A total of 2.7×10¹³ viral particles was injected pereye.

An earlier version of the L-opsin coding rAAV2/5 used in previousunsuccessful experiments contained an extra 146 base pair segmentbetween the splice donor/acceptor site and the translational start codonof the L-opsin gene that had been carried over from the cloning vector.Because we were concerned that this fragment may have interfered withtransgene expression, a second version of L-opsin rAAV2/5 in which theextra 146 by had been removed was used in later experiments describedhere. In addition to modifying the vector, we also increased the amountof viral particles delivered per eye by approximately 16-fold, from1.7×10¹² to 2.7×10¹³. Thus, we cannot conclude from this set ofexperiments what exact titer of viral particles was required to producethe effects on color vision behaviour, or exactly what effects, if any,the extra 146 bp had on transgene expression in earlier unsuccessfulattempts.

The single-stranded DNA genome of conventional rAAV vectors, includingrAAV2/5 used here, is devoid of Rep coding sequences. Thus, the vectorgenome is stabilized predominantly in an episomal form; however, thepotential for integration exists²¹. According to NIH guidelines, theviral vector used here is rated biosafety level 1 (BSL1), and animalbiosafety level 1(ABSL1) meaning no special precautions were required inhandling the virus or animals treated with the virus. Followingtreatment, squirrel monkeys had an increase in AAV antibody titers,ranging from 4-12 fold. Antibody titers remained unchanged in untreatedcontrol animals who were housed with treated animals.

Subretinal Injections. Subretinal injections were performed by avitreo-retinal surgeon (T. B. C.) using a KDS model 210 syringe pumpunder a stereomicroscope. A 500 μL Hamilton Gastight (#1750TTL) LuerLock syringe was connected to 88.9 cm of 30 gauge teflon tubing withmale Luer Lock adapters at both ends (Hamilton 30TF double hub), whichwas then connected to a 30 gauge Becton Dickinson Yale regular bevelcannula (ref #511258) that was manually bent to produce a 135° angle 1.5mm from the tip. All components were sterilized prior to use. Thesyringe and tubing were filled with sterile lactated Ringers solution toproduce a dead volume of approximately 210 μL. Just prior to injection,300 μL of rAAV was withdrawn using a rate of 100 μL/min.

Squirrel monkeys were anesthetized using intramuscular injections ofketamine (15 mg/kg) and xylazine (2 mg/kg); atropine (0.05 mg/kg) wasalso given to reduce airway secretions. The eye was dilated with 2-3drops of tropicamide (1%) and treated with 1 drop each of betadine (5%),vigamox (0.5%), and proparacaine (1%). Subconjunctival injection of 0.1mL of lidocaine (2%) was given and the anterior portion of the eye wasexposed by performing a temporal canthotomy followed by limitedconjuntival peritomy. Eyelids were held open with a speculum designedfor premature infants. A temporal sclerotomy was made 1 mm posterior tothe limbus with a 27 gauge needle, through which the injection cannulawas inserted. Three subsequent 100 μL injections were made at differentsubretinal locations using an infusion rate of 1060 μL/min.Post-procedure, 0.05 mL each of decadron (10 mg/mL), kenalog (40 mg/mL),and cephazolin (100 mg/mL) were injected subconjunctivaly; 1 drop eachof betadine (5%) and vigamox (0.5%) and a 0.6 cm strip of tobradex (0.3%tobramycin, 0.1% dexamethasone) ointment were applied topically; 10-20mL of subcutaneous fluids (sterile lactated Ringers) were also given.Subsequent administration of steroids and analgesics were administeredas needed post-procedure for potential inflammation or discomfort.

Confocal Microscopy. The animal in FIG. 1 c and d succumbed torespiratory illness, unrelated to gene therapy, approximately 2 yearsand 3 months post-injection. The retina was fixed in 4% paraformaldehydein phosphate buffered saline (PBS), and rinsed in PBS with 10% and 30%sucrose. It was sequentially incubated with 10% Normal Donkey Serum,rabbit monoclonal antibody to M/L opsin (Chemicon AB5405), and a Cy3(red) conjugated donkey anti-rabbit antibody (Jackson Immunoresearch).Confocal images were analyzed using ImageJ (rsbweb.nih.gov). In themiddle panel of FIG. 1 e, magenta dots mark cone locations, and the redanti-M/L-opsin antibody staining was removed to show GFP-expressing(green) cells more clearly.

Behavioural Colour Vision Assessment. A three-alternative forced-choiceparadigm in which position and saturation of the stimulus was randomizedbetween trials was used. Monkeys had to discriminate the location of acoloured patch of dots that varied in size and brightness, surrounded bysimilarly varying grey dots. When animals touched the coloured target, apositive tone sounded and a juice reward was given; the next stimulusappeared immediately. (The squirrel monkey shown in FIG. 2 c is drinkinga reward from a previous trial.) If the wrong position was chosen, anegative tone sounded, and a 2-3 sec “penalty time” occurred before thenext trial.

For each hue, monkeys were tested on up to 11 different saturationsranging from 0.01 to 0.11 in u′, v′ colour space (CIE 1976) and athreshold was calculated, which was taken as the saturation required toreach a criterion of 57% correct, the value determined to besignificantly greater than chance (33% correct, P=0.05); see ref 6 forfull details.

REFERENCE FOR EXAMPLE 1

-   1. Wiesel, T. N. & Hubel, D. H. Single-cell responses in striate    cortex of kittens deprived of vision in one eye. J. Neurophysiol.    26, 1003-1017 (1963).-   2. Jacobs, G. H. A perspective on color vision in platyrrhine    monkeys. Vision Res. 38, 3307-3313 (1998).-   3. Li, Q., Timmers, A. M., Guy, J., Pang, J. & Hauswirth, W. W.    Cone-specific expression using a human red opsin promoter in    recombinant AAV. Vision Res. 48(2007).-   4. Reffin, J. P., Astell, S. & Mollon, J. D. Trials of a    computer-controlled colour vision test that preserves the advantages    of pseudo-isochromatic plates. in Colour Vision Deficiencies X69-76    (Kluwer Academic Publishers, Dordrecht, 1991).-   5. Regan, B. C., Reffin, J. P. & Mollon, J. D. Luminance noise and    the rapid determination of discrimination ellipses in colour    deficiency. Vision Res. 34, 1279-1299 (1994).-   6. Mancuso, K., Neitz, M. & Neitz, J. An adaptation of the Cambridge    Colour Test for use with animals. Vis. Neurosci. (2006).-   7. Kuchenbecker, J., Sahay, M., Tait, D. M., Neitz, M. & Neitz, J.    Topography of the long- to middle-wavelength sensitive cone ratio in    the human retina assessed with a wide-field color multifocal    electroretinogram. Vis. Neurosci. 25, 301-306 (2008).-   8. Mancuso, K. et al. Recombinant adeno-associated virus targets    passenger gene expression to cones in primate retina. J. Opt. Soc.    Am. A Opt. Image Sci. Vis. 24, 1411-1416 (2007).-   9. Nathans, J., Piantanida, T. P., Eddy, R. L., Shows, T. B. &    Hogness, D. S. Molecular genetics of inherited variation in human    color vision. Science 232, 203-210 (1986).-   10. Shapley, R. Specificity of cone connections in the retina and    color vision. Focus on “Specificity of cone inputs to macaque    retinal ganglion cells”. J. Neurophysiol. 95, 587-588 (2006).-   11. DeValois, R. L. & DeValois, K. K. A multi-stage color model.    Vision Res. 33, 1053-1065 (1993).-   12. Jacobs, G. H., Williams, G. A., Cahill, H. & Nathans, J.    Emergence of Novel Color Vision in Mice Engineered to Express a    Human Cone Photopigment. Science 315, 1723-1725 (2007).-   13. Makous, W. Comment on “Emergence of Novel Color Vision in Mice    Engineered to Express a Human Cone Photopigment”. Science 318, 196    (2007).-   14. Maguire, A. M. et al. Safety and efficacy of gene transfer for    Leber's congenital amaurosis. N. Engl. J. Med. 358, 2240-2248    (2008).-   15. Bainbridge, J. W. & Ali, R. R. Success in sight: The eyes have    it! Ocular gene therapy trials for LCA look promising. Gene Ther.    15, 1191-1192 (2008).-   16. Cideciyan, A. V. et al. Human gene therapy for RPE65 isomerase    deficiency activates the retinoid cycle of vision but with slow rod    kinetics. Proc. Natl. Acad. Sci. U. S. A. 105, 15112-15117 (2008).-   17. Wang, Y. et al. A locus control region adjacent to the human red    and green visual pigment genes. Neuron 9, 429-440 (1992).-   18. Mauck, M. C., Mauncuso, K., Kuchenbecher, J., Connor, T. B.,    Hauswirth, W. W., Neitz, J., Neitz, M. . Longitudinal evaluation of    expression of virally delivered transgenes in gerbil cone    photoreceptors. Vis. Neurosci. 25, 273-282 (2008).-   19. Nathans, J., Thomas, D. & Hogness, D. S. Molecular genetics of    human color vision: the genes encoding blue, green, and red    pigments. Science 232, 193-202 (1986).-   20. Neitz, M., Neitz, J. & Jacobs, G. H. Spectral tuning of pigments    underlying red-green color vision. Science 252, 971-974 (1991).-   21. Büning, H., Perabo, L., Coutelle, O., Quadt-Humme, S. &    Hallek, M. Recent developments in adeno-associated virus vector    technology. J. Gene Med. 10, 717-733 (2008).

EXAMPLE 2 Description and Validation of New LED-Based, Wide-Field, Colormf-ERG

The electroretinogram (ERG) is an electrophysiologic recording techniqueused to measure electrical activity of the entire retina. Theelectrochemical potential of retinal cells change in response to light,which in turn induces voltage on an electrode placed on the corneaand/or sclera. The first ERGs were recorded by a Swedish physiologistworking in the mid-1800's in amphibian retina. Since this time thetechnique has been widely incorporated in clinical practice as adiagnostic tool. Marriage of physiology to engineering has led to avariety of stimuli and analysis paradigms which can tease out specificcellular responses or region specific information. The latter has beenmotivated by the fact that the topographical organization of the retinaplays an important role in disease with different diseases beingcharacterized by different affected retinal areas. Early attempts toevaluate the function of specific retinal regions using the ERGilluminated only a small patch of retina, however, such “focal ERGs”have the drawback that light reflected from the focal area onto otherretinal regions produces an ERG response that cannot be uncoupled fromthat produced by the region of interest. The other problem is thatobtaining any type of a topographical map of retinal function using themono-focal approach is prohibited by the time required to obtain ERGsserially from many different retinal regions. Both problems have beensolved with the development of the multifocal- (mf-) ERG, pioneered byErich Sutter in the early 1990s (Sutter, 1991). Mf-ERGs perform a seriesof individual focal ERG experiments simultaneously by taking advantageof either (1) correlation techniques or (2) frequency encoding. In thisway, a complete topographical map of electrical responses over a largeretinal region is obtained in a relatively short period of time.

The typical ERG apparatus for mf-ERGs stimulates a hexagonal patch ofretina with a 20 degree radius and uses video display. More recently adisplay that employs white light emitting diodes (LEDs) was designed foruse with a frequency encoding method for obtaining mf-ERGs. Having astimulus that could reach further into the periphery would be useful inthe early detection of retinal diseases that progress from peripheral tocentral retina. Additionally, individuals with normal trichromatic colorvision express three distinct photopigments, or opsins, in separateclasses of cone photoreceptor: short- (S-), middle- (M-), and long- (L-)wavelength sensitive. The S-cones are maximally sensitive to shortwavelengths of light near 420 nm; M-cones have their maximal responsenear 530 nm; and the L-cones are most sensitive near 560 nm. Thus, anmf-ERG stimulus containing LEDs of different wavelengths would haveapplications in characterizing the topography of expression of thedifferent opsin transgenes in the eyes of living subjects treated withgene therapy.

In particular, in gene therapy treatments administering recombinantadeno-associated virus (rAAV) carrying a human L-opsin gene M-opsin geneor S-opsin gene to primates that have two cone types to producetrichromatic color vision. As such, a non-invasive objective method isneeded to determine the locations of functioning opsin expression invivo.

Here we describe a wide-field color mf-ERG capable of stimulating aradius greater than 70 degrees. The wide field mf-ERG has a coloredLED-based stimulus that incorporates a new design capable of maintainingviable signal-to-noise ratio (SNR) out into the far peripheral retina.

mf-ERG and Stimulus Panel

An LEDs as a light source was chosen because new advancements in LEDtechnology allow for a large number of focused photons to be emittedfrom a point source. Additionally, LEDs are available in a variety ofsingle peak narrow-bandwidth packages. The stimulus contained 1024doublet pixels each containing a red (653 nm, half-bandwidth 22 nm, FIG.2.1c) and a green (527 nm, half-bandwidth 33 nm, FIG. 2.1d) LED. Thus,the new display had 2,048 paired green and red LEDs. LEDs have inherentmanufacturing variations in their breakdown resistance. Since the amountof current is proportional to the number of photons per unit time,applying a constant voltage across the LEDs would result in varyingphoton output. To circumvent these issues and ensure repeatability andlinearity, constant current integrated circuit chips (Allegro A6276)were used to drive the LEDs (FIG. 2.1 b). These devices are designed tomaintain constant current despite fluctuations in anode voltage. Toprevent variation in peak wavelength over varied intensities, a pulsefrequency modulation (PFM) signal was used (Swanson, Ueno, Smith, &Pokorny, 1987).

Using red and green LEDs, it is possible to isolate responses of L- orM-cones using silent substitution. Integrating the spectral compositionof the green LED with the spectral sensitivity curves of the human M-and L- photopigments (FIG. 2.1 e) yields that approximately equal quantaare caught by both photopigments (FIG. 2.1 f). In contrast, the red LEDis six times more effective at stimulating the L photopigment than it isthe M (FIG. 2.1 f). Isolation of responses that are due to transductionfrom an introduced L-opsin transgene, M-opsin transgene or S-opsintransgene are straightforward in primates because red, blue or greenLEDs can be chosen that are much more effective for the L-opsin M-opsinor S-opsin transgene respectively than for the endogenous pigments ofthe untreated dichromatic primates. In the case of Squirrel monkeys,they have S-cones maximally sensitive near 430 nm, and they can expressany of several variants of middle-to-long wavelength opsin including Lor M. The monkey used in validation experiments had M-cones sensitivenear 532 nm, in addition to his S-cones. Thus, following theadministration of the L-opsin transgene via subretinal injection, themf-ERGs of squirrel monkey were predicted to show elevations in mf-ERGamplitude to red light in regions corresponding areas of the retinatransduced, and distal areas would show progressively lower amplitudesto the L-cone isolating stimulus.

Resolution of the stimulator was 1024 LED over a larger stimulating areaof about 150 degrees of visual field. Sensitivity of the measurement canbe increased by summing more retinal responses per unit area and byusing ultra-bright LEDs. Other new techniques that were used to preventperipheral SNR fall-off included the use of trapezoidal shaped printedcircuit boards that when placed edge-to-edge created a geodesic dome(FIG. 2.1 a). The advantage of this design was that LEDs were heldequidistantly from and pointing toward a single focal point where thesubject's pupil was positioned. Any variation in directionality causedby imperfect sphericity was corrected by aiming each LED individually.In the typical usage, LEDs from areas of the dome shaped stimulator weresummed so that there were 37 individual segments, which togethersubtended the 150° of visual angle, thus allowing a wide-fieldfunctional map of an area covering almost the entire retina to beproduced.

There are two mathematical methodologies available to code and decodethe topographical regions on the recording electrode: One is across-correlation technique called m-sequence and the other is afrequency encoding technique called cyclic summation. Cyclic summationis preferred in our application because it has been empirically shown toprovide higher signal-to-noise ratio than m-sequence (Lindenberg, Horn,& Korth, 2003). Also, cyclic summation cannot be done using any kind ofconventional CRT or LCD video display. Cyclic summation requiresindependent control of every segment of the display. Conventional videodisplays update the entire screen with every video frame typically at 60Hz. In cyclic summation, each segment being analyzed is updating at aslightly different frequency. Typically, the “center frequency” is 30 Hzbut each segment fractionally differenent from 30 Hz, i.e., 30.00 Hz,30.10 Hz, 30.20 . . . etc. In the analysis, responses to different areasof the retina to different segment frequencies are used to generate amap of retinal responses which are read out as electrophysiological“activity.” For example, after gene therapy using an L opsin gene in aprimate retina containing only M and S cones, areas of retina thatexpress the newly introduced L opsin will have higher electricalactivity in response to the red LED light relative to the green lightthan areas of the retina not expressing the transgene. This allows theeffectiveness of the gene therapy in terms of areas responding theirtime course to be monitored with an objective measure.

In practice, we define the following parameters; f_(c)=center frequency,T=total time, Q=number of segments, and n=0. . . (Q-1). The number ofcycles per segment is given by

$\begin{matrix}{{cycles}_{n} = {{f_{c} \cdot T} - {\left\lbrack {\frac{Q - 1}{2} + n} \right\rbrack.}}} & (2.1)\end{matrix}$

Then, f_(n) represents the frequencies at which each segment is encodedinto the stimulus is

$\begin{matrix}{f_{n} = {\frac{{cycles}_{n}}{T}.}} & (2.2)\end{matrix}$

Finally, by windowing and summing the recorded retinal waveform, definedas w(t), region specific signal, Activity_(n), can be extracted inequation 2.3,

$\begin{matrix}{{Activity}_{n} = {\sum\limits_{n = 0}^{({Q - 1})}{\sum\limits_{m = 0}^{({{cycles}_{n} - 1})}{{w\left( {\frac{m}{f_{n}}\mspace{14mu} \ldots \mspace{14mu} \frac{\left( {m + 1} \right)}{f_{n}}} \right)}.}}}} & (2.3)\end{matrix}$

For our purposes, f_(c)=30 Hz, T=40 seconds, and Q=37. In the retina, anadditional advantage of the cyclic summation method are that theintensity and temporal frequency of the LEDs can be specified to isolatecone photoreceptor responses and silence rod photoreceptor responses.

Repeatability, Linearity, and SNR

Measurements for repeatability and linearity were made using a UDT S370Optometer (UDT Instruments, San Diego, Calif.). LED outputs weremeasured in microwatts (uW) at seven different intensity settings, inrandom order. Three complete sets of data were taken on three separatedays. All measurements were taken after the instrument becameequilibrated with the ambient room temperature, which reflects normaloperation of the instrument.

Signal-to-noise ratio was measured using a human subject by firstplacing an opaque material in front of the stimulus and runningsuccessive mf-ERGs. Voltages received during the blocked trials weretaken as the noise of the instrument. Signal was then measured byperforming mf-ERGs on four human subjects with normal trichromatic colorvision. To compare SNR as a function of eccentricity, signal and noisewere broken into different eccentric rings. Best subject and the averageof all subjects were calculated for each ring. Tests involving humansubjects were done in accordance with the principles embodied in theDeclaration of Helsinki

Viral Vector and Subretinal Injections

To validate whether the instrument operated as expected, a mixture oftwo recombinant adeno-associated viruses was injected sub-retinally in agerbil (Meriones unguiculatus) and a squirrel monkey (Saimiri sciureus).One virus, rAAV.CHOPS2053.GFP, carried a gene for green fluorescentprotein (GFP) and the other virus, rAAV.CHOPS2053.RHLOPS, coded forhuman L-opsin. The L-opsin virus was identical to the GFP virus exceptthat a Not I restriction fragment containing the coding sequence forgreen fluorescent protein was replaced with a 1.2 kb Not I restrictionfragment containing recombinant human L opsin cDNA. The opsin geneencoded a human L pigment that is predicted from the deduced amino acidsequence to be maximally sensitive to 562 nm light. In order to providea method for visualizing transduced cone photoreceptors usingimmunohistochemistry in future experiments, the sequence of the human Lopsin transgene was changed so that the last 12 amino acids matched theknown epitope for the monoclonal antibody OS-2. This antibody waspreviously shown to specifically label S or UV cones in mammalian andprimate retina. The C-terminal 12 amino acids of human S opsin weredemonstrated to be the epitope. The substitution of the C-terminal 12amino acids of S-opsin into L-opsin is not predicted to change thespectral sensitivity of the L-opsin trans-gene product.

Subretinal injections were performed. Briefly, gerbils were anesthetizedusing a combination of Ketamine (50 mg/kg) and Xylazine (2 mg/kg), andit received two 5 uL subretinal injections of a 1:1 (volume:volume)mixture of rAAV.CHOPS2053.GFP and rAAV.CHOPS2053.RHLOPS that were placedin the superior retinal area. A color mf-ERG was then performed on thisanimal at 6 months post-injection. Squirrel monkeys was anesthetizedusing 15 mg/kg ketamine and 2 mg/kg xylazine, and they received two 100uL subretinal injections of a virus mixture containing 110 uL ofrAAV.CHOPS2053.GFP and 220 uL of rAAV.CHOPS2053.RHLOPS. Both injectionswere positioned near the fovea, with the first injection placedsuperiorly, and the second injection placed in the inferotemporal regionof the retina. A color mf-ERG was performed on this animal about 42weeks, or 10.5 months post-injection. All of these procedures wereconducted in accordance with the experimental animal care and usageguidelines of the United States National Institutes of Health.

Fundus Exams

Both the gerbil and squirrel monkey had fundus images taken at multipletime points post-injection to observe expression of the GFP transgeneover time. Fundus images were obtained using the fluorescein angiographymode of the RetCam II digital imaging system (Massie Laboratories,Pleasanton, Calif. For the gerbil, images were taken with a lensdesigned for detecting retinopathy in premature infants, which providesa 130° field of view, and for the monkey, a high magnification 30° lenswas used. Thus, multiple fundus images from the squirrel monkey werepieced together into a montage, in order to show a comparable retinalarea.

Results

Measures of repeatability, linearity, and signal-to-noise ratio (SNR)were performed to evaluate the wide-field color ERG system. Moreproperly, given n=2 . . . 8, m=½^(n), and t=[0, ˜3.5, 7] days, thenintensity, I_(m)(t), is said to be repeatable if

$\begin{matrix}{s = {{3 \cdot \sqrt{\frac{1}{2}{\sum\limits_{t = 1}^{3}\left( {{I_{m}(t)} - \overset{\_}{I_{m}(t)}} \right)^{2}}}} \leq ɛ}} & (2.4)\end{matrix}$

where ε is defined as

ε=0.05·max(photon_(m)).   (2.5)

The instrument was said to be linear if Pearson's R² was greater than0.95. And finally, signal (in SNR) was is calculated by averagingresponse from four subjects, and noise was taken as the residual signalwhile an opaque material blocked stimulus.

Repeatability and linearity are demonstrated in FIG. 2.2. Each datapoint is an average of optometer measurements for the particularintensity setting taken on three separate days; error bars are thestandard deviations with a 99.7% confidence interval. Pearson's Rcorrelation was calculated and the coefficient of determination (R²)values is shown. The red LEDs shared 0.9987 total variance and the greenLEDs shared 0.9996 total variance demonstrating system is linear andrepeatable over time. SNR values are shown in Table 2.1. The SNR washigh for the inner segments of the stimulus panel; it decreased in moreperipheral regions but remained high enough to allow measurements intothe far periphery.

Because the GFP-coding virus and the L-opsin-coding virus were injectedtogether at the same locations, fluorescence fundus images could beregistered with mf-ERG data from the same animal in order to validatethe redesigned wide-field color mf-ERG system. Areas of high GFPexpression corresponded well to areas of high mf-ERG voltage in responseto the L-cone isolating red stimulus (FIG. 2.3). Animals wereinsensitive to the red 653 nm wave-length light prior to injection,verifying that the signal measured in animals post-injection is truesignal coming from the introduced L-opsin transgene product. In thesquirrel monkey, the red-light mf-ERG data showed high voltage in theregions that corresponded to the fluorescence fundus images.

To evaluate whether the concave surface of the newly developedstimulator produces ERG amplitudes that are relatively constant withretinal eccentricity an experiment was performed in which flicker ERGresponses were measured under two different conditions. In FIG. 2.4, thecircles represent ERG responses from an LED traveling down a linear pathwhile the subject fixated forward. On the same graph, trianglesrepresent ERG responses from an LED traveling on a rotating boom whilethe subject fixated forward. The boom held the LEDs perpendicular to thecornea. Results from this experiment demonstrate the increase in signalgiven by the convex stimulator, compared to a traditional flat-panel LCDscreen or CRT monitor. The curved stimulus design, in addition to theuse of ultra-bright LEDs as the light source, increased the SNRsufficiently high in the far periphery to ensure viable signals wererecorded from cone photoreceptors.

In the conventional mf-ERG, the signal amplitudes are greatly reduced inthe periphery because the illumination from a flat screen falls offroughly as the cosine with increasing eccentricity. In our design, theredesigned stimulus had concave structure holding the LEDs such that theinner surface pointed perpendicular to the stimulated retinal area. Inaddition, similar to traditional mf-ERG stimulators, the number of LEDsin each segment was increased with eccentricity to compensate for thedecrease in cone density.

If 40% of cones express the transgene the increase in red sensitivityshould be that expected from a spectral ERG in which 20% of the totalERG contribution is from L opsin while 80% is from M opsin. This isconsistent with the observed red sensitivity in both monkeys andgerbils.

This is the first time that measurements from cones in the farperipheral retina have been achieved. Results from these experimentsindicate that the wide-field color mf-ERG system is a valid techniquefor measuring the topography of opsin expression in living subjects, andit will serve as an important tool for evaluating success of genetherapy in humans.

REFERENCES FOR EXAMPLE 2

-   Sutter, E. E. (1991). The Fast m-Transform: A Fast Computation of    Cross-Correlations with Binary m-Sequences. SIAM Journal on    Computing, 20(4), 686-694.-   Swanson, W. H., Ueno, T., Smith, V. C., & Pokorny, J. (1987).    Temporal modulation sensitivity and pulse-detection thresholds for    chromatic and luminance perturbations. Journal of the Optical    Society of America A—Optics, Image Science and Vision, 4(10), 13.-   Lindenberg, T., Horn, F. K., & Korth, M. (2003). Cyclic summation    versus m-sequence technique in the multifocal ERG. Graefes Archive    of Clinical Experimental Ophthalmology., 241(6), 505-510.

1. A method for cone cell gene therapy in a primate, comprisingadministering to the eye of a primate in need of cone cell gene therapya recombinant gene delivery vector comprising (a) a promoter region,wherein the promoter region is specific for retinal cone cells; and (b)a gene encoding a therapeutic, wherein the gene is operatively linked tothe promoter region; wherein in vivo expression of the therapeutic incone cells of the primate serves to treat the primate in need of conecell gene therapy.
 2. The method of claim 1, wherein the gene therapyserves to treat a cone cell disorder.
 3. The method of claim 2, whereinthe cone cell disorder is selected from the group consisting of colorblindness, blue cone monochomacy, achromatopsia, incompleteachromatopsia, rod-cone degeneration, retinitis pigmentosa (RP), maculardegeneration, cone dystrophy, blindness, Stargardt's Disease, andLeber's congenital amaurosis.
 4. The method of 1 wherein the primate isof the Parvorder Catarrhini.
 5. The method of claim 1 wherein thepromoter comprises a sequence selected from the group consisting of theL opsin promoter (SEQ ID NO: 1), the M opsin promoter (SEQ ID NO: 2),and the S opsin promoter (SEQ ID NO: 3),
 6. The method of claim 1,wherein the gene delivery vector further comprises an enhancer elementupstream of the promoter, wherein the gene is operatively linked to theenhancer element.
 7. The method of claim 6, the enhancer element isspecific for primate retinal cone cells.
 8. The method of claim 7,wherein the enhancer element comprises the nucleic acid sequence of SEQID NO:
 51. 9. The method of claim 1, wherein the gene delivery vectorfurther comprises an intron comprising a splice donor/acceptor region,wherein the intron is located downstream of the promoter region and islocated upstream of the gene.
 10. The method of claim 1, wherein thegene encodes a therapeutic protein comprising a polypeptide selectedfrom the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ IDNO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:49, and a polymorph of SEQ ID NO: 11 selected from the group consistingof: (i) Thr65Ile (ii) Ile111Val (iii) Ser116Tyr (iv) Leu153Met (v)Ile171Val (vi) Ala174Val (vii) Ile178Val (viii) Ser180Ala (ix) Ile230Thr(x) Ala233Ser (xi) Val236Met (xii) Ile274Val (xiii) Phe275Leu (xiv)Tyr277Phe (xv) Val279Phe (xvi) Thr285Ala (xvii) Pro298Ala; and (xviii)Tyr309Phe.
 11. The method of claim 1, wherein the gene delivery vectorcomprises a recombinant adeno-associated virus (AAV) gene deliveryvector.
 12. The method of claim 11, wherein the AAV gene vectorcomprises rAAV2/5 vector.
 13. The method of claim 1, wherein the methodrestores visual capacity in the primate.
 14. The method of claim 1,wherein the primate suffers from color blindness, and the primate isable to visualize new colors as a result of the therapy.
 15. The methodof claim 14, wherein the promoter comprises a sequence selected from thegroup consisting of L opsin promoter (SEQ ID NO: 1), M opsin promoter(SEQ ID NO: 2), and S opsin promoter (SEQ ID NO: 3); and the geneencodes one or more polypeptides comprising a sequence selected from thegroup consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11; and apolymorph of SEQ ID NO: 11 selected from the group consisting of (i)Thr65Ile (ii) Ile111Val (iii) Ser116Tyr (iv) Leu153Met (v) Ile171Val(vi) Ala174Val (vii) Ile178Val (viii) Ser180Ala (ix) Ile230Thr (x)Ala233Ser (xi) Va1236Met (xii) Ile274Val (xiii) Phe275Leu (xiv)Tyr277Phe (xv) Va1279Phe (xvi) Thr285Ala (xvii) Pro298Ala; and (xviii)Tyr309Phe.
 16. The method of claim 1, wherein the primate has a visiondisorder in which its photoreceptors are healthy.
 17. The method ofclaim 1, wherein the primate is an adult primate.
 18. An isolatednucleic acid expression vector comprising: (a) a promoter region,wherein the promoter region is specific for primate retinal cone cells;and (b) a gene encoding a therapeutic, wherein the gene is operativelylinked to the promoter region.
 19. The isolated nucleic acid expressionvector of claim 18, wherein the promoter comprises a nucleic acidselected from the group consisting of L opsin promoter (SEQ ID NO: 1), Mopsin promoter (SEQ ID NO: 2), and S opsin promoter (SEQ ID NO: 3). 20.The isolated nucleic acid expression vector of claim 18, wherein theisolated nucleic acid expression vector further comprises an enhancerelement upstream of the promoter, wherein the gene is operatively linkedto the enhancer element.
 21. The isolated nucleic acid expression vectorof claim 20, wherein the enhancer element comprises a nucleic acidsequence according to SEQ ID NO:
 51. 22. The isolated nucleic acidexpression vector of claim 18, further comprising an intron comprising asplice donor/acceptor region, wherein the intron is located downstreamof the promoter region and is located upstream of the gene.
 23. Theisolated nucleic acid expression vector of claim 18, wherein the geneencodes a therapeutic protein selected from the group consisting of SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and a polymorph of SEQ ID NO:11 selected from the group consisting of: (i) Thr65Ile (ii) Ile111Val(iii) Ser116Tyr (iv) Leu153Met (v) Ile171Val (vi) Ala174Val (vii)Ile178Val (viii) Ser180Ala (ix) Ile230Thr (x) Ala233Ser (xi) Val236Met(xii) Ile274Val (xiii) Phe275Leu (xiv) Tyr277Phe (xv) Val1279Phe (xvi)Thr285Ala (xvii) Pro298Ala; and (xviii) Tyr309Phe.
 24. The isolatednucleic acid expression vector of claim 18, wherein the vector comprisesan AAV vector.
 25. A formulation comprising packaged viral particlescomprising the nucleic acid expression vectors of claim
 18. 26. Arecombinant host cells transfected or transduced with the nucleic acidexpression vector of claim
 18. 27. A wide field, color multi-focalelectroretinogram (mf-ERG) comprising: (a) an electroretinogram (ERG)comprising (i) a recording electrode that is (A) designed for placementon at least one of a cornea and a sclera of at least one eye of asubject and (B) arranged to output at least one signal generated by theat least one eye; and (ii) a computing system communicatively coupled tothe recording electrode, the computing system comprising (A) at leastone processor and (B) data storage containing instructions executable bythe at least one processor to carry out a set of functions, the set offunctions including processing and saving the at least one signalgenerated by the at least one eye; (b) a retinal stimulator comprisingmatched light sources selected from the group consisting of red, green,blue, and ultraviolet light sources, wherein the matched light sourcesare connected to the ERG and in operation can be independently frequencymodulated at rates between about 1 Hz and about 60 Hz, inclusive,wherein the stimulator in operation is capable of stimulating a retinalfield of a subject throughout an operating radius of at least about 70degrees; (c) one or more constant current integrated circuit chipsarranged to drive the stimulator; and (d) a pulse-frequency modulatorconnected to the retinal stimulator, wherein in operation thepulse-frequency modulator is capable of controlling individualstimulator segments while keeping relative spectral content of the lightconstant.
 28. The mf-ERG of claim 27, wherein the matched light sourcesare paired red and green light sources.
 29. The mf-ERG of claim 27,wherein the matched light sources are triplets of red, green, and bluelight sources.
 30. The mf-ERG of claim 27, wherein the matched lightsources are quartets of red, green, blue, and ultraviolet light sources.31. The mf-ERG of claim 30, wherein the matched light sources comprisematched light emitting diodes (LEDs).
 32. The mf-ERG of claim 27,wherein the retinal stimulator comprises a concave surface comprising aseries of trapezoidal-shaped circuit boards placed edge-to-edge, whereinthe concave surface positions the matched light sources so in operationthey are held equidistantly from and pointing toward a single focalpoint where a subject's pupil can be positioned.
 33. The mf-ERG of claim32, wherein the concave surface comprises a geodesic dome.
 34. Themf-ERG of claim 27, wherein the set of functions further comprisescoding and decoding topographical regions on the recording electrodeusing a cyclic summation technique.
 35. The mf-ERG of claim 27, whereinthe ERG further comprises an amplifier; and wherein the computing systemis communicatively coupled to the amplifier.
 36. A method fordetermining a location of functioning opsin expression in a subject,comprising use of the mf-ERG of claim 27, wherein the recordingelectrode is placed on at least one of a cornea and a sclera of at leastone eye of a subject; stimulation of the subject's retinal field withthe retinal stimulator; and determining responses of different areas ofthe subject's retina to different stimulation frequencies to generate amap of retinal responses, wherein the map provides a location offunctioning opsin expression in a subject.
 37. (canceled)